Project Summary While mitochondrial dysfunction is evident in the failing heart, its precise role in disease progression is unclear and the mechanism(s) of its origin is not well understood. Mitochondria play a key role in many cellular processes, including oxidative phosphorylation, metabolite synthesis and calcium storage. They are also important regulators of cell death and monitor changes in the intracellular environment such as presence of reactive oxygen species and DNA damage. The BCL-2 proteins play a key role in regulating mitochondrial membrane permeabilization and apoptosis. We recently discovered that the anti-apoptotic BCL-2 protein MCL-1 is critical for normal mitochondrial function and cardiac homeostasis. Loss of MCL-1 in cardiac myocytes leads to rapid mitochondrial dysfunction, development of heart failure, and early mortality. Surprisingly, MCL-1 deficient myocytes display signs of necrosis rather than apoptosis as would be expected, suggesting that besides its anti- apoptotic role, MCL-1 has an essential but yet unidentified role in maintaining mitochondrial function in cardiac myocytes. We have also found that MCL-1 exists both in the outer mitochondrial membrane (MCL-1OM) and in the mitochondrial matrix (MCL-1Matrix) in the heart. While a study has implicated MCL-1OM in regulating apoptosis, the function of MCL-1Matrix is still unknown. Based on our preliminary data, we propose to study the hypothesis that MCL-1 has a dual role in maintaining cardiac mitochondrial function and health that is dependent on its mitochondrial location: MCL-1OM facilitates mitochondrial fission and mitophagy of aberrant mitochondria to prevent activation of unnecessary apoptosis, whereas MCL-1Matrix promotes mitochondrial fusion to preserve bioenergetic capacity and protect against autophagosomal degradation during nutrient limiting conditions. This hypothesis will be tested with three specific aims. In Specific Aim 1, we will characterize the role of MCL-1Matrix in regulating mitochondrial fusion, function, turnover and survival. In Specific Aim 2, we will delineate the role of MCL-1OM in regulating mitochondrial fission and turnover. We will determine if MCL-1OM interacts with Drp1 to promote asymmetrical fission and removal of damaged mitochondria and whether this is part of its pro-survival function. Finally, in Specific Aim #3, we will investigate whether MCL-1OM also functions as a receptor for LC3 to drive selective degradation of mitochondrial by autophagosomes. We will utilize both isolated cardiac myocytes and genetically modified mouse models combined with proteomics, cell and molecular biology to uncover the bi- functional roles of MCL-1OM and MCL-1Matrix in myocytes under baseline conditions and in response to challenge (fasting and myocardial infarction). These studies will provide important new insights into the relationship between mitochondrial dynamics, turnover and survival in the heart. A better understanding of how mitochondrial function is regulated in the heart under normal and disease conditions such as myocardial infarct will contribute towards future clinical management of heart disease.